Novel polymerizable fatty acids, phospholipids and polymerized liposomes therefrom

ABSTRACT

The invention relates to an oral drug delivery system which delivers biologically active substances to the mucosal tissue of the intestine utilizing novel polymerized liposomes. Novel polymerizable fatty acids having a polymerizable group, a surfactant group, and a functional group, and optionally coupled to ligands which target mucosal tissue in the intestine are disclosed. Novel negatively charged polymerizable lipids which have phosphatidyl inositol (PI), phosphatidyl glycerol (PG) or phosphatidyl serine (PS) groups on a polymerizable backbone are also described.

INTRODUCTION

[0001] The present invention relates to novel polymerizable fatty acidsand phospholipids useful for preparing polymerizable liposomes for oraland/or mucosal delivery of vaccines, allergens, diagnostics andtherapeutics. In particular, the present invention relates topolymerizable fatty acids having a polymerizable group, a surfactantgroup, and a functional group, such as octadecadienoyl-polyethyleneglycol-succinic acid (ODPEGSu) compounds, and polymerizable liposomesprepared therefrom. The present invention further relates topolymerizable fatty acids coupled to targeting ligands with an affinityfor human and mammalian intestinal M cells and similar cells in thenasopharyngeal cavity, such as lectins or proteins or peptides which canbind to M cells, and to polymerizable liposomes incorporating them. Theinvention also relates to negatively charged polymerizable lipids,specifically derivatives of polymerizable liposomes which havephosphatidyl inositol (PI), phosphatidyl glycerol (PG) or phosphatidylserine (PS) groups on a polymerizable backbone, and to liposomesprepared therefrom. The invention still further relates to the use ofthe polymerized liposomes of the present invention as, or in,pharmaceutical compositions for oral delivery of a variety of diagnosticor therapeutic agents, including drugs, allergens and vaccines. Theliposomes of the present invention provide increased stability in thegastrointestinal (G-I) tract, and increased flexibility in targetingliposomes to particular cells to enhance the uptake of encapsulatedtherapeutic agents.

BACKGROUND OF THE INVENTION

[0002] Drug Delivery

[0003] Drug delivery takes a variety of forms, depending on the agent tobe delivered and the administration route. The most convenient way toadminister drugs into the body is by oral administration. However, manydrugs, in particular proteins and peptides, are poorly absorbed andunstable during passage through the gastrointestinal (G-I) tract. Theadministration of these drugs is generally performed through parenteralinjection.

[0004] Although oral vaccination is more convenient, vaccines aregenerally given through injection. This is particularly true with killedor peptidic vaccines, because of their low absorbability and instabilityin the G-I tract. A problem with systemic immunization is that it maynot effectively induce mucosal immune responses, particularly productionof IgA, that are important as the first defense barrier to invadedmicroorganisms. For this reason, it would be beneficial to provide oralvaccination, if the problems of low absorbability and instability couldbe overcome.

[0005] Controlled release systems for drug delivery are often designedto administer drugs to specific areas of the body. In thegastrointestinal tract it is important that the drug not be eliminatedbefore it has had a chance to exert a localized effect or to pass intothe bloodstream.

[0006] Enteric coated formulations have been widely used for many yearsto protect drugs administered orally, as well as to delay release.Several microsphere formulations have been proposed as a means for oraldrug delivery. For example, PCT/US90/06433 by Enzytech discloses the useof a hydrophobic protein, such as zein, to form microparticles; U.S.Pat. No. 4,976,968 to Steiner et al. discloses the use of “proteinoids”to form microparticles; and European Patent Application 0,333,523 by theUAB Research Foundation and Southern Research Institute discloses theuse of synthetic polymers such as polylactic acid-glycolic acid to formmicrospheres.

[0007] Particles less than ten microns in diameter, such as themicroparticles of EPA 0,333,523, can be taken up by cells in specializedareas, such as Peyer's patches and other intestinal mucosal lymphoidaggregates, located in the intestine, especially in the ileum, into thelymphatic circulation. Entrapping a drug or antigen in amicroparticulate system can protect the drug or antigen from acidic andenzymatic degradation, yet still allow the drug or antigen to beadministered orally, where they are taken up by the specialized uptakesystems, and release the entrapped material in a sustained manner or areprocessed by phagocytic cells such as macrophages. When the entrappedmaterial is a drug, elimination of the first-pass effect (metabolism bythe liver) is highly advantageous.

[0008] Liposomes

[0009] Conventional liposomes have been proposed for use as an oral drugdelivery system, for example, by Patel and Ryman, FEBS Letters 62(1),60-63 (1976). Liposomes are typically less than 10 microns in diameter,and, if they were stable to passage through the G-I tract, may beabsorbed through Peyer's patches (Aramaki, Y., H. Tomizawa, T. Hara, K.Yachi, H. Kikuchi, and S. Tsuchiya, 1993 Stability of liposomes in vitroand their uptake by rat Peyer's patches following oral administration.Pharm. Res. 10:1338, 1331; Childers, N., F. R. Donya, N. F. Magoo, andS. M. Michalek 1990. Ultrastructural study of liposome uptake by M cellsof rat Peyer's patch: an oral vaccine system for delivery of purifiedantigen. Regional Immunology 3:8-16). Liposomes also have some featuresthat should be advantageous for a particulate system for oral drug orantigen delivery. The phospholipid bilayer membrane of liposomesseparates and protects entrapped materials in the inner aqueous corefrom the outside. Both water-soluble and -insoluble substances can beentrapped in different compartments, the aqueous core and bilayermembrane, respectively, of the same liposome. Chemical and physicalinteraction of these substances can be eliminated because the substancesare in these different compartments. Further, liposomes are easy toprepare. However, liposomes are physically and chemically unstable, andrapidly leak entrapped material and degrade the vesicle structure.Without fortifying the liposomes, they are not good candidates for oraldrug or antigen delivery. Thus, despite the early proposal for use ofconventional liposomes in oral drug delivery, their use has still notbeen accepted.

[0010] Several methods have been tried to fortify liposomes. Somemethods involve intercalating cholesterol into the bilayer membrane orgenerating the liposomes using phospholipids with high meltingtemperature or physically stabilizing preformed liposomes withexcipients such as simple sugars or polysaccharides. Generally, thesemethods are not believed to be sufficient in making liposomes for oraldelivery since during oral delivery liposomes are exposed to an acidicPh in the stomach and bile salts and phospholipases in the intestine.These conditions typically dissolve the characteristic liposomal bilayermembrane and contents are released and degraded.

[0011] Polymerized Liposomes

[0012] Polymerization of liposomes has been shown in vitro to be aneffective means of stabilizing the liposomes and reducing problems ofdegradation, agglomeration, and leakage of encapsulated drugs.Polymerized liposomes have been developed in attempts to improve oraldelivery of encapsulated drugs (Chen et al., WO 9503035). The ability ofpolymerized liposomes to survive the G-I tract has also beeninvestigated (Chen et al., 1995, Proceed. Internat. Symp. Control. Rel.Bioact. Mater. 22; Chen et al., 1995 Proc. 3rd U.S. Japan Symposium onDrug Delivery Systems; Brey, R. N., 1997, Proc. 4th U.S. Japan Symposiumon Drug Delivery).

[0013] A number of compounds have been reported to form polymerizedliposomes. For example, U.S. Pat. No. 4,248,829 discloses phospholipidscontaining di-yne acyl chains polymerizable by ultraviolet light toyield intermolecular or intramolecular cross-linking.

[0014] U.S. Pat. No. 4,485,045 discloses polymerizable phosphatidylcholine derivatives containing an unsaturated lower aliphatic acyloxylonger chain alkanoyloxy moiety. The polymerizable site in thephosphatidyl choline derivatives is a terminal ethylene group on theacyloxy substituent.

[0015] U.S. Pat. No. 4,808,480 discloses heterocyclic compoundscontaining disulfide bonds that are used to form polymerizablephospholipids. The phospholipids incorporate the heterocyclic disulfidecompounds as terminal substituents on the glyceryl acyl groups, andpolymerize upon ring-opening of the heterocyclic substituents.

[0016] U.S. Pat. No. 4,594,193 discloses polymerizable lipid compoundscontaining mercaptan groups. These lipids polymerize by formation ofintermolecular disulfide linkages.

[0017] U.S. Pat. No. 5,160,740 discloses polymerization of apolymerizable 2,4-diene phospholipid, cholesterol, and a polymerizable2,4-diene fatty acid to form a polymerized macromolecular endoplasmicreticulum. The reticulum is reported to be stable in surfactantsolutions and capable of enclosing hemoglobin.

[0018] U.S. Pat. No. 5,466,467 discloses derivatives of phosphatidylcholine containing polymerizable acyl chain moieties and metal-chelatinggroups. The phospholipids contain iminodiacetic acid covalently bondedto the choline in the polar head group. Cross linked phospholipidmembranes generated from monomeric units can be used to immobilizeenzymes and proteins on the surface of the liposomes via metal bridges.

[0019] Further, U.S. Pat. No. 5,366,881 discloses phosphatidyl cholinederivatives containing different polymerizable groups positioned atvarious sites in the acyl chains to achieve altered membrane fluidityproperties. Additionally, the mixture of non-polymerizable phospholipidswith polymerizable phospholipids provides for bilayer liposomes capableof conditional release of encapsulated material.

[0020] A number of additional polymerizable phospholipids are describedin Regen, in Liposomes: from Biophysics to Therapeutics (Ostro, ed.,1987), Marcel Dekker, N.Y. Additional polymerizable moieties containedwithin the acyl chains of phospholipids or within the polar head grouphave been described and are found in Singh, A., and J. M. Schnur, 1993,“Polymerizable Phospholipids”, in Phospholipids Handbook, Gregor Cevc,ed., Marcel Dekker, New York. Various other polymerizable phospholipidsand fatty acids have been described, having methacrylate, vinylbenzene,diacetylenes, and azidoformaloxy groups within the structure of the acylchains.

[0021] Although polymerized liposomes, generally, are more stable thantheir unpolymerized counterparts, it is not clear that the improvedstability thus far achieved is by itself sufficient to enable theseliposomes to deliver effective doses of drugs administered orally.Recent studies have investigated the possibility of modifyingpolymerized liposomes to contain a molecule or ligand which selectivelytargets M cells and other absorptive cells in the mammalian intestine(Chen et al., 1996, Pharmaceutical Research 13:1378-1383). Incorporationof a targeting ligand is believed to increase the adhesion efficiency ofthe modified polymerized liposome on M cell surfaces, and thus toincrease the efficiency of absorption of drugs encapsulated in thoseliposomes. M cells are specialized epithelial cells dispersed within thefollicle associated epithelium (FAE) overlying the Peyer's patches inmammalian small intestine. The use of targeting ligands specific forsurface receptors on M cells, enterocytes or other cells requires newchemistries to effectively incorporate such ligands without compromisingthe stability or safety of the polymerized liposome.

[0022] A variety of methods have been described for covalently couplingof bioactive ligands to the surface of conventional liposomes. U.S. Pat.No. 5,171,578 discloses the chemical coupling of the glycoproteinstreptavidin to the surface of liposomes via a modified phosphatidylethanolamine. Because of selective binding affinity to biotin, suchsurface modified liposomes can be used to directly bind biotinylatedproteins to their surface.

[0023] U.S. Pat. No. 5,204,096 describes the covalent coupling ofpeptides to the surface of liposomes by activating peptides withcarbodiimide followed by coupling to active carboxyl groups exposed onthe surface of liposomes. In this case, surface carboxyl groups areprovided by the inclusion of aminoalkanes, such as stearylamine ordiamino alkanes in the lipid bilayer.

[0024] U.S. Pat. No. 5,258,499 discloses the preparation of a liposomecytokine complex in which the procedure for covalent attachment ofreceptor-binding interleukin-2 involves treatment of the cytokine withsuccinimidyl-4-(p-maleimidophenyl)butyrate as a linker followed bylinkage to activated liposome surfaces. In this case, the activatedliposome surface consists of phosphatidyl ethanolamine modified withsuccinimidyl-S-acetylthioacetate.

[0025] Zalipsky et al (Zalipsky, S., Mullah, N., Harding, J. A.,Gittelman, J., Guo, L. and DeFrees, S. A., 1997, Bioconjug. Chem.8:111-118) described the synthesis of a lipid anchor for the surfacemodification of liposomes, containing distearoylphosphatidylethanolamine(DSPE) as a lipid anchor, heterobifunctional polyethylene glycol (PEG)with a molecular weight of 2000 as a linking moiety, and biological celladhesive ligand [YIGSR peptide or Sialyl Lewis (X) oligosaccharide(SLX)]. Allen et al (Allen, T. M., Brandeis, E., Hansen, C. B. Kao, G.Y. Zalipsky, S. 1995. Biochim Biophys Acta. 1237:99-108) described thederivitization of the surface of sterically stabilized liposomes. Thepolyethylene glycol (PEG)-lipid derivativepyridylthiopropionoylamino-PEG-distearoylphosphatidylethanolamine(PDP-PEG-DSPE) was synthesized and incorporated into liposomes.Thiolysis of the PDP groups resulted in formation of reactive thiolgroups on the liposome surface which reacted with maleimide-activatedantibodies to yield covalent attachment of the antibodies. Kirpotin etal (Kirpotin, D., Park, J. M., Hong, K., Zalipsky, S., Li, W. L.,Carter, P., Benz, C. C., Papahadjopoulos, D. 1997. Biochemistry36:66-75) described the formation of liposomes conjugated viaPEG-modified distearoylphosphatidyl phosphatidylethanolamine to Fabfragments of a humanized recombinant Mab against the extracellulardomain of the breast cancer marker HER2/neu by maleimide-terminatedmembrane-anchored spacers of two kinds for covalent attachment at thedistal terminus of the PEG chain.

[0026] Lectin Targeting of Liposomes

[0027] Lectins have been proposed as promising moieties to use astargeting ligands. Lectins are a broad group of proteins, usuallyglycoproteins of plant origin, with binding specificity for particularcarbohydrates. Like any targeting ligand, lectins can be covalentlybound to the lipids of the liposome, or can be non-covalently attachedto the liposome by a combination of short-range intermolecular forcesand simple steric entanglement. Surface-bound lectins can aid in theselective targeting of liposomes with entrapped drug or antigen tocarbohydrate counter-ligands expressed on cell receptors or othersurface glycoproteins. In order to effectively target particular cells,the lectins must be attached to the liposome so that the site-specificportion of the lectin is exposed and available for binding to cells.Additionally, the targeting lectin must be incorporated into theliposome in a manner that does not destabilize the liposome or allowphysical release of the targeting ligand from the liposome surface. Awide variety of lectins with selectivity to intestinal absorptive cellsand M cells has been identified (Gianasca, P. J., K. T. Gianasca, P.Falk, J. I. Gordon, and M. R. Neutra 1994. Gastrointen. Liver Physiol.30:G1108-1121; Clark, M. A., M. A. Jepson, and B. H. Hirst 1995. Lectinbinding defines and differentiates M-cells in mouse small intestine andcaecum. Histochem Cell Biol. 104:161-168). Recent work has shown thatlectins with selectivity to intestinal M cells and enterocytes can beincorporated into liposome bilayers and the liposomes subsequentlypolymerized (Chen et al., 1996, Pharmaceutical Research 13:1378-83).These lectin-modified polymerized liposomes show increased efficacy intargeting liposomes to Peyer's patches in the G-I tract. However, thelectin-modified anchoring lipids used in these studies were notstructurally optimized for stability within the polymerized liposomebilayer and formed patches of non-polymerized lipids that contributed toinstability.

[0028] Despite the advances in liposome technology and drug delivery,there remains a need for stable and efficacious polymerized liposomes,and new polymerizable compounds that can be incorporated intopolymerizable liposomes to improve stability, binding selectivity, andefficiency of drug delivery. There additionally remains a need for newprocesses to manufacture polymerizable liposomes incorporating targetingmolecules or ligands, and to manufacture polymerizable liposomes whichencapsulate drugs.

SUMMARY OF THE INVENTION

[0029] The present invention encompasses novel chemical compounds usefulfor the preparation of polymerizable liposomes, and polymerizableliposomes made therefrom. The novel chemical compounds can be used tocreate polymerizable liposomes with improved properties, includingenhanced stability in the G-I tract, increased ability to targetspecific cells, and ability to effectively deliver therapeutics anddiagnostics and other agents orally or mucosally. The invention isparticularly suited for the delivery of oral vaccines.

[0030] The present invention encompasses, in one embodiment, novelpolymerizable fatty acids comprising a polymerizable group such as 2,4octadecadienoyl (2,4OD), a surfactant group such as polyethylene glycol(PEG), and an acid functional group such as succinic acid (Su) andchemically stable linkages between the groups. The polymerizable groupcan contain a variety of polymerizable moieties such as double bonds,triple bonds, or thiol groups. In a particularly preferred embodiment,the polymerizable fatty acids have the formula:

CH₃—(CH₂)₁₂—CH═CH—CH═CH—C(O)—(OCH₂CH₂)_(n)—O—C(O)—CH₂—CH₂—CO₂H

[0031] where n is an average number of monomer units, determined by theaverage molecular weight of the polyethylene reagent used. The molecularweight ranges from about 200 to about 2000 g/mol.

[0032] The present invention also relates to polymerizable fatty acidswhich have been coupled to a ligand, such as a lectin, or moleculecapable of targeting intestinal M-cells and absorptive enterocytes andsimilar cells lining the nasopharyngeal cavity.

[0033] The present invention also relates to polymerized liposomes whichincorporate the polymerizable fatty acids, either as coupled to atargeting ligand such as a lectin, or in a non-derivatized form. Thesepolymerized liposomes are obtained, inter alia, by polymerizing amixture of a polymerizable lipid and a polymerizable fatty acid or apolymerizable fatty acid coupled to a targeting ligand, and optionallyother non-polymerizable phospholipids and/or cholesterol.

[0034] The present invention also relates to polymerizable lipids whichcontain a negatively-charged hydrophilic group, such as phosphatidylinositol (PI), phosphatidyl glycerol (PG) or phosphatidyl serine (PS).These polymerizable lipids can be based on a glyceryl backbone such as1,2-di(2,4-octadecadienoyl) or other polymerizable lipid backbone.

[0035] Definitions

[0036] As used herein, the term “liposome” is defined as an aqueouscompartment enclosed by a lipid bilayer. (Stryer, Biochemistry, 2dEdition, W. H. Freeman & Co., p. 213 (1981)). The liposomes can beprepared by a thin film hydration technique followed by a fewfreeze-thaw cycles which are known in the art. Liposomal suspensions canalso be prepared according to methods known to those skilled in the art,for example, as described in U.S. Pat. No. 4,522,811, which isincorporated herein by reference in its entirety.

[0037] As used herein, the term “polymerized liposome” is defined as aliposome in which some, most or all of the constituent phospholipids arecovalently bonded to each other by inter and/or intra molecularinteractions. The phospholipids can be bound together within a singlelayer of the phospholipid bilayer (the leaflets) and/or bound togetherbetween the two layers of the bilayer.

[0038] As used herein, the term “conventional liposome” refers to anunpolymerized liposome.

[0039] The degree of polymerization in the polymerized liposomes canrange from 30 to 100 percent; i.e., up to 100 percent of the availablebonds are formed. The size range of polymerized liposomes is betweenapproximately 15 nm to 10 μm. The polymerized liposomes can be loadedwith up to 100% of the material to be delivered, when the material ishydrophobic and attracted by the phospholipid layers. In general, about5 to about 40 percent of the material is encapsulated when the materialis hydrophilic.

[0040] As used herein, the term “trap ratio” is defined as the ratio ofinner aqueous phase volume to total aqueous phase volume used.

[0041] As used herein, the term “radical initiator” is defined as achemical which initiates free-radical polymerization.

[0042] As used herein, the term “reverse phase evaporation technique” isdefined as a method involving dissolving a lipid in an organic solvent,adding a buffer solution, and evaporating the organic solvent at reducedpressure, as described by Szoka, F. Jr., and Papahadjopoulos, D., Proc.Natl. Acad. Sci. USA, Volume 75, No. 9, pp. 4194-4198 (1978).

[0043] As used herein, the term “freeze-thaw technique,” or “F-T,” isdefined as freezing a suspension in a cryogenic fluid such as liquidnitrogen, and subsequently thawing the suspension in a roughly 30° C.water bath.

[0044] As used herein, the terms “mucosa” or “mucosal tissue” refers toa epithelial tissue, such as intestinal lamina propria, a layer ofsmooth muscle in the digestive tract, nasopharyngeal epithelial tissue,lung epithelial tissue, ocular epithelial tissue, or vaginal epithelialtissue. Usually, these tissues are protected by a layer of mucous, acomplex mixture of mucin and other proteins up to 200 μm thick, whichserves as a mechanical barrier against microbial pathogens and as amilieu for secreted effectors such as secretory immunoglobulins. Mucosaldelivery as used herein is meant to include delivery through bronchi,gingival, lingual, buccal, nasal, oral, vaginal and intestinal mucosaltissue.

[0045] As used herein, the term “buffer solution” is defined as anaqueous solution or aqueous solution containing less than 25% of amiscible organic solvent, in which a buffer has been added to controlthe Ph of the solution. Examples of suitable buffers include but are notlimited to PBS (phosphate buffered saline), TRIS(tris-(hydroxymethyl)aminomethane), HEPES (hydroxyethylpiperidine ethanesulfonic acid), and TES(2-[(tris-hydroxymethyl)methyl]amino-1-ethanesulfonic acid).

[0046] As used herein, the term “leaflets” is defined as a single layerof phospholipids in the bilayer forming the liposome.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The invention relates to an oral or mucosal drug delivery systemto deliver drugs to the mucosal tissue of the intestine and otherepithelial surfaces which utilizes polymerized liposomes as the drugcarriers. More specifically, the invention relates to novelpolymerizable fatty acids, novel polymerizable phospholipids andliposomes made therefrom. The polymerizable fatty acids andphospholipids are used to prepare liposomes with significant stabilityin the G-I tract. Further, the polymerizable fatty acids andphospholipids can be used to covalently attach targeting molecules tothe liposomes, such as lectins which target M cells in intestinalPeyer's patches, absorptive intestinal enterocytes and other similarcells in other mucosal surfaces. The polymerizable fatty acids are usedto improve the preparation and loading of the polymerized liposomes. Thepolymerized liposomes prepared using these novel fatty acids orphospholipids are especially useful for targeting the delivery ofvaccines and antigens to M cells. The following describes the novelfatty acids and phospholipids, how they are prepared and how they can beutilized to prepare stable polymerized liposomes.

[0048] Polymerizable Fatty Acids

[0049] The present invention encompasses, in one embodiment, novelpolymerizable fatty acids which can be used both to increase thestability of polymerized liposomes incorporating them, and to provide afunctional acid linking group to conveniently, efficiently andeffectively attach targeting ligands to polymerized liposomes. Thepolymerizable fatty acids comprise at one end a polymerizable group, atthe other end an acid functional group, and a surfactant group, betweenthe polymerizable and functional groups, forming the central portion ofthe fatty acid, and optionally chemically stable linking moietiesbetween these groups. It is preferred that the functional group be anacid functional group.

[0050] For example, the structure of these novel fatty acids in oneembodiment is:

R₄—X-PEG-Y—B

[0051] wherein R₄, the polymerizable group, is a lipophilic chain (fattyacid chain) with at least one polymerizable functional group that willenable polymerization; X or Y are independently a functional linkagesuch as an ester bond, an ether bond, an amide bond or a carbamate; B isan acid functionality, —NH₂, or an aldehyde; and PEG is the preferredsurfactant group which can vary in molecular weight as described below.

[0052] The structure of these novel fatty acids gives them uniquefunctionality and particular utility when used in conjunction withpolymerizable liposomes. The polymerizable group allows the novel fattyacid molecules to co-polymerize with polymerizable phospholipids in apolymerizable liposome, so that the molecules are covalently bound tothe polymerized liposome, rather than attached in a less-stable fashion,such as by intercalation or steric entanglement. The functional acidgroup provides a convenient reaction site which can be derivatized usingknown techniques to attach any targeting ligand capable of bonding tothe acid or derivatized acid moiety. The surfactant group is disposedbetween the polymerizable group and the functional acid group, andcomprises a polymeric chain with hydrophilic and hydrophobic regions.

[0053] The surfactant group serves several functional purposes. Thelength of the polymeric chain of the surfactant group can be chosen tobe short or long, and the relative hydrophilicity/hydrophobicity of thechain can be altered, depending on the desired properties of theliposome. The polymeric chain should not be long enough to affect theability of the lipophilic moiety to participate in the lipid packing. Along-chain surfactant group with significant hydrophilicity, forexample, can extend away from the liposome into the surroundingsolution, providing the liposome with numerous hydrophilic “hairs”protecting the liposome body and effectively “disguising” it to aid itspassage through the G-I tract. A short-chain surfactant group with lesshydrophilicity will stay closer to the body of the liposome, and willtend to coil and tangle, to give the liposome numerous hydrophilic coilsor tangles close to the liposome surface. It will be appreciated thatseveral configurations can be achieved, by varying the length andhydrophilicity of the polymer chain. When the fatty acid is coupled to atargeting ligand and incorporated into a polymerized liposome, thepolymer chain of the surfactant group additionally serves as a “spacer”between the liposome and the targeting group, allowing the targetinggroup to be held closer or farther from the body of the liposome, asdesired.

[0054] The polymerizable group can be any group capable of coupling tothe surfactant group and co-polymerizing with polymerizablephospholipids. A wide variety of polymerizable groups are suitable, andit will be appreciated that the particular choice of polymerizablegroups will depend upon the polymerizable phospholipid and surfactantgroups chosen. For example, it is convenient to use a mono-, di- orpoly-unsaturated aliphatic carboxylic acid, which can polymerize with apolymerizable phospholipid through the double or triple bond or bonds,and can couple to hydroxy-terminated surfactant groups through the acidmoiety. Specific examples of polymerizable groups include, but are notlimited to, unsaturated aliphatic acid groups such asCH₃(CH₂)_(m)CH═CH—CH═CHCOOH where the number of methylene groups (m) canvary from 4 to 12. The double bonds or polymerizable functionalities canbe anywhere in the chain so long as they provide an environment suitablefor polymerization and packing. One or more of such functionalities canbe present in a molecule.

[0055] The surfactant group comprises a polymeric chain with hydrophilicand hydrophobic regions, capable of coupling to both the polymerizablegroup and the functional acid group. Polyethers such as polyethyleneglycol, polypropylene glycol, and their copolymers, for example, aresuitable surfactant groups. Poly(lactic acid) may also be used. Apreferred surfactant group is polyethylene glycol, as it is readilycoupled to the preferred enoic polymerizable groups and the preferreddioic functional acid groups discussed below.

[0056] The functional group can be an acid capable of coupling to thesurfactant group. Diacids are preferred, as they are easily attached tothe preferred polyether surfactant groups. Particularly preferred aresaturated, aliphatic diacids of the formula:

HO—C(O)—(CH₂)_(b)—C(O)—OH

[0057] where b is an integer from 0 (i.e., oxalic acid) to 12,preferably 0 to 4. Unsaturated diacids having from 2 to 14 carbon atomsare also suitable. For convenient coupling to the surfactant group,diacids which can be used in their anhydride form are especiallypreferred, such as succinic acid (succinic anhydride). Alternatively,the functional group can be an amine, an amide or diamine.

[0058] In a preferred embodiment, the polymerizable group is a2,4-dienoyl, the surfactant is a polyethylene glycol group (PEG), andthe functional group is a short-chain diacid acid group. In thisembodiment, the fatty acids have the formula:

CH₃—(CH₂)_(a)—CH═CH—CH═CH—C(O)—(OCH₂CH₂)_(n)—O—C(O)—(CH₂)_(b)—CO₂H

[0059] where a is an integer from 0 to 18, preferably 4 to 12, b is aninteger from 0 to 12, preferably 0 to 4 and the value of n depends onthe average molecular weight of the polyethylene glycol reagent used tosynthesize the fatty acids; n can range from about 4 (PEG-200) to about45 (PEG-2000). It will be appreciated that n is an average value, notgenerally integral, which characterizes a mixture of chain lengthspresent in commercially available polyethylene glycols of a particularmolecular weight average.

[0060] In a more preferred embodiment, the polymerizable group is an 2,4octadecadienoyl group (2,4OD), the surfactant is a polyethylene glycolgroup (PEG), and the functional acid group is a succinic acid group(Su). In this particularly preferred embodiment, the fatty acids havethe formula

CH₃—(CH₂)₁₂—CH═CH—CH═CH—C(O)—(OCH₂CH₂)_(n)—O—C(O)—CH₂—CH₂—CO₂H

[0061] where n is about 8.7, corresponding to the average n in PEG-400.A polyethylene glycol of any desired molecular weight can beincorporated into the 2,4-ODPEGSu fatty acid. For use with the targetingligands described below, however, polyethylene glycols with averagemolecular weights from about 200 to about 2000 are preferred, and amolecular weight average of about 400 or about 1900 is most preferred.These preferred ODPEGSu polymerizable fatty acids can be formed by firstreacting 2,4-octadecadienoic acid with a desired molecular weightpolyethylene glycol to form 2,4-ODPEG, then derivatizing the ODPEGproduct with succinic anhydride to form ODPEGSu. For a detailedsynthesis, see the Examples infra.

[0062] Targeted Polymerizable Fatty Acids

[0063] The novel polymerizable fatty acids of the present invention areeasily derivatized and covalently linked to ligands capable of targetingparticular cells in the G-I tract. The fatty acids are first coupled toa targeting ligand, then copolymerized with a polymerizable liposome, toform a stable, polymerized liposome with the desired targeting ligandcovalently attached. The polymerization can be carried out in thepresence of a desired therapeutic agent, such as a vaccine or antigen,or the polymerized liposome can be loaded with a therapeutic agent afterpolymerization, using known techniques. The resulting stable, targetedliposome can be used to effectively and selectively deliver therapeuticagents to M cells in the G-I tract. The targeted fatty acids of thepresent invention are thus especially useful for the oral delivery oftherapeutic agents, such as vaccines.

[0064] The present invention encompasses the novel fatty acids describedabove, which have been further derivatized (if necessary) and coupled toa ligand or molecule capable of targeting particular cells in thegastrointestinal tract. It is expected that liposomes prepared withthese targeted ligands will selectively bind to the targeted cells,thereby increasing the effectiveness of delivery of encapsulated drugs.The targeting ligand can be, for example, a lectin with an affinity forhuman and mammalian intestinal M cells. Preferred ligands are lectinssuch as EEA (Euonymus Europaeus), a fluorescently labelled EEA, FITC-EEA(fluorescein isothiocyante-EEA), UEA-I (Ulex Europaeus Agglutinin I),and WGA (Wheat Germ Agglutinin). Many other ligands are potentiallyuseful for mucosal application; for example, class II framework Mab,(Estrada, A., M. R. McDermott, B. J. Underdown, and D. P. Snider. 1995.Intestinal immunization of mice with antigen conjugated to anti-MHCclass II antibodies. Vaccine. 13:901-907), ICAM-1, or any protein orpeptide ligand that selective binds to M cells including alkalinephosphatase, bacterial surface proteins (Jones, B. D., N. Ghouri, and S.Falkow. 1994. Salmonella typhimurium initiates murine infection bypenetrating and destroying the specialized epithelial M cells of thePeyer's patches. J. Exp. Med. 180:15-23) or viral proteins (Amerongen,H., G. A. R. Wilson, B. N. Fields, and M. R. Neutra. 1994. Proteolyticprocessing of reovirus is required for adherence of intestinal M cells.J. Virol. 68:8428-8432) or ligands or antibodies having affinity formucosal addressin cell adhesion molecule-1 (Madcam-1) (Sampaio, S. O.,X. Li, M. Takeuchi, C. Mei, U. Francke, E. C. Butcher, and M. J.Briskin, 1995. Organization, regulatory sequences, and alternativelyspliced transcripts of the mucosal addressin cell adhesion molecule-1 J.Immunol. 155:2477-2486).

[0065] In a preferred embodiment, the targeting ligand is a lectin suchas ERA, and the fatty acid is a dienoyl-polyethylene glycol-diacidderivative, such as ODPEGSu. The coupling reaction to covalently attacha lectin to a fatty acid such as ODPEGSu can be carried out using thetechniques known in the art such as that described in Chen et al., 1996,Pharmaceutical Research 13:1378-1383. The coupling of 2,4-ODPEGSu withEEA is shown in the Examples, infra.

[0066] The amount of targeting ligand used in the polymerizable liposomewill depend on the specific target. In general, the ratio of targetingligand to polymerizable material is about 10-100 molecules per liposomes(about 1/10,000 to about 1/1000 w/w).

[0067] Polymerized Liposomes Using Polymerizable Fatty Acids

[0068] The novel polymerizable fatty acids of the present invention canbe used to form an oral drug delivery system to deliver drugs to themucosal tissue of the intestine. The present invention thus encompassespolymerized liposomes which incorporate these novel polymerizable fattyacids, and the use of the polymerized liposomes as drug carriers. Thefatty acids can be used either in their non-derivatized form, to enhancethe stability of the polymerized liposomes, or coupled to a ligand whichtargets particular cells in the G-I tract, as described above. Thepolymerized liposomes are obtained by polymerizing a mixture of apolymerizable lipid and a polymerizable fatty acid or polymerizablepolymer-coupled fatty acid or polymerizable targeted fatty acid of thepresent invention, using conventional liposome polymerizationtechniques, such as irradiation, redox initiation, radical initiation,and the like.

[0069] The polymerizable lipids used in conjunction with thepolymerizable fatty acids and targeted polymerizable fatty acids of thepresent invention are not limited to any particular lipids. Any lipidcan be used which is polymerizable and is capable of forming polymerizedliposomes. A wide variety of polymerizable lipids have been described inthe literature; see, e.g., Regen, in Liposomes: From Biophysics toTherapeutics (Ostro, ed., 1987), Marcel Dekker, N.Y., and Singh andSchnur, Polymerizable Phospholipids, in Phospholipids Handbook, 1993,Marcel Dekker, New York which are incorporated herein by reference.Preferred polymerizable lipids include diene containing phospholipidswith uncharged head groups, such as glycerol, inositol, or serine, orcharged head groups such as choline or ethanolamine. A particularlypreferred polymerizable lipid is1,2-di(2,4-octadecadienoyl)-3-phosphatidylcholine (DODPC).

[0070] The polymerizable fatty acids and targeted polymerized fattyacids can be any of the species described herein. Without being bound byany particular theory, it is believed that the 2,4-ODPEGSu fatty acidco-polymerizes with the polymerizable lipid, and that the hydrophilictail of 2,4-ODPEGSu incorporated into the liposome extends away from theliposome surface and into any surrounding aqueous phase. The PEG chainthus enhances the stability of the liposome by creating a stericallystabilized liposome, in which the liposome body is somewhat protected bythe protruding and entangled copolymerized ODPEGSu chains. Highermolecular weight polyethylene glycols (i.e., average molecular weightabove about 2000) may destabilize the liposome, while lower molecularweight PEGs (i.e., average molecular weights from about 200 to 2000)will have a net stabilizing effect. The resulting polymerized liposomesthus have increased stability in the G-I tract and ability to passthrough mucus layer, and additionally can be targeted to particularcells of the intestine when targeted polymerizable fatty acids are used.

[0071] When targeted fatty acids are used, the polymer chain of thesurfactant group additionally serves as a spacer between the liposomeand the targeting ligand attached to the fatty acid. Accordingly, themolecular weight of the polyethylene glycol should be chosen in order toachieve the desired spacing, while still allowing the fatty acid tocopolymerize with the lipids. The lower molecular weight polyethyleneglycols are thus believed to be more suitable; i.e., those with averagemolecular weights of about 200 to about 2000, preferably about 200 toabout 1500, and most preferably about 400.

[0072] The polymerized liposomes of the present invention canadditionally contain non-polymerizable compounds, so long as the amountsof polymerizable lipids and polymerizable fatty acids or targetedpolymerizable fatty acids are sufficient to give the resultingpolymerized liposomes adequate stability. For example, non-polymerizablefatty acids or non-polymerizable phospholipids known in the art and usedfor conventional liposome formation may be used. In addition,cholesterol can be used for added stability. A preferrednon-polymerizable compound is cholesterol which can be included in molarratios of up to 50% with the polymerizable components.

[0073] The polymerized liposomes of the present invention may beutilized for the delivery of a wide variety of compounds, includingvaccines, antigens, allergens and other therapeutic agents ordiagnostics. They have particular utility in the oral and/or mucosaldelivery of vaccines and antigen release devices. For example, thepolymerized liposomes of the present invention may be designed to carrya wide variety of antigens including, but not limited to, diphtheriatoxoid, influenza hemeagglutinin, ospA antigen from Lyme diseasebacterium, and HTLV envelope protein antigen. Antigens to poliovirus,rhinovirus, rabies, vaccinia, Epstein-Barr virus, hepatitis, HTLV,herpes virus and human immunodeficiency virus are just examples of themany types of antigens which may be encapsulated into the liposomes ofthe present invention. They may also be utilized for the oral deliveryof a wide variety of therapeutics, including but not limited to,chemotherapy agents, antibiotics, insulin, cytokines, interferon,hormones, calcitonin, hormones, fertility drugs, antiviral agents (ddI,AZT, ddc, acyclovir and the like), antibacterial agents, antifungalagents, DNA and RNA nucleotides.

[0074] Negatively Charged Polymerizable Lipids

[0075] For still greater flexibility and utility in creating oral drugdelivery systems, the present invention also encompasses novelpolymerizable phospholipids with negatively charged groups.Incorporating negatively charged groups into a polymerized liposomegreatly expands the use of the liposomes by taking advantage of thedesirable properties of liposomes while additionally utilizing theelectrostatic charge to improve and enhance the ability of the liposomesto entrap therapeutic agents. The resulting negatively chargedpolymerizable liposomes have superior trap ratios, and thus areespecially effective in delivering the entrapped therapeutic agents.

[0076] The negatively charged polymerizable lipids of the presentinvention include polymerizable lipids which have phosphatidyl inositol(PI), phosphatidyl glycerol (PG) or phosphatidyl serine (PS) groups on apolymerizable backbone. These polymerizable lipids can be used to createpolymerizable liposomes incorporating the negatively-charged PI, PG orPS groups, using conventional techniques. Because of capacity tointeract with divalent cations, such negatively charged polymerizablephospholipids can assume alternate configurations in aqueoussuspensions. In the presence of metal ions, for example, Ca²⁺ or Mg²⁺ions, jelly-roll or cochelate structures can be formed; these structuresconsist of tightly packed bilayer membranes in which water has beensqueezed out of the internal spaces. By harvesting such structures bycentrifugation, followed optionally by lyophilization, and exposure todivalent metal-ion chelating agents, such as EGTA or EDTA, in thepresence of drug or protein to be encaptured, a high and reproducibledegree of loading of typically configured spherical bilayer liposomescan be obtained.

[0077] The negatively charged polymerizable lipids have the structure:

[0078] wherein at least one of R, R′ or R″ is independently phosphorylinositol, phosphoryl glycerol or phosphoryl serine and at least one ofthe remaining two groups is a polymerizable group, consisting of acylchains containing dienoic acids, diacetylenic acids, methacrylate sidegroups, acrylates, or thiol or disulfide containing acids. For example,the negatively charged polymerizable lipids may also have the followingstructure:

[0079] where X is glycerol, inositol or serine; and

[0080] R₁ and R₂ are independently a polymerizable group selected fromthe group consisting of a diene group, a diacetylene group, amethacrylate group, and a thiol group. The polymerizable group ispreferably a hydrocarbon chain containing one or more of theabove-mentioned polymerizable moieties. The hydrocarbon chain can befrom C₄ to C₃₀ and higher if desired. Although any polymerizablebackbone can be used with these negatively charged polymerizable lipids,a particularly preferred backbone is 2,4-DODPC. Thus, a preferrednegatively charged polymerizable lipid is

[0081]  in which R is phosphoryl inositol, phosphoryl glycerolphosphoryl or serine.

[0082] These novel negatively charged polymerizable lipids can besynthesized according to the methods described in Confurius and Zwaal,Biochimin Biophysica Acta, 488:36-42 (1977) wherein polymerizable PG, PIor PS may be synthesized by a transphosphatidylation catalyzed byphospholipase D in the presence of protected glycerol, inositol orserine followed by a deprotection step. For example, DODPC is dissolvedin diethyl ether (distilled from P₂O₅ to remove trace of alcohol) at aconcentration of 20 mg/ml. L-serine is first lyophilized from a 10%(w/v) aqueous solution to remove trace of methanol and is subsequentlydissolved at 45° C. at different concentrations up to saturation (46%w/v) in 100 mM acetate buffer (Ph 5.6) containing 100 mM CaCl₂.Phospholipase D is added to the serine solution at 45° C. to a finalconcentration of 1 IU/ml. An equal volume of the DODPC solution in etheris added and the incubation flask is immediately closed, in order toavoid ether evaporation. Incubation is carried out at 45° C. withstirring to complete mixing of both phases. Usually, two additionalportions of phospholipase D equal to the starting amount are added after30 minutes and 60 minutes respectively. Incubation is stopped after 90minutes by addition of 100 mM EDTA (equivalent to two volumes of acetatebuffer). Ether is evaporated at room temperature under a stream ofnitrogen gas and the aqueous layer is mixed with 4.3 vol. ofchloroform/methanol (5.8 v/v) and is stirred for 30 min. The singlephase mixture is filtered through a glass filter G-2 and the filtrate isstirred for 10 min. with 1 volume of water and 3.7 volumes ofchloroform. After centrifugation (10 min, 3000×g) the lower chloroformlayer is collected and mixed with an equal volume of absolute ethanol,followed by evaporation to dryness under reduced pressure. The residueis dissolved in chloroform. Similar incubation is carried out at 37° C.in which serine is replaced by ethanolamine, glycerol, methanol, orethanol in order to establish optimal conditions leading to the highestyields of DODPS.

[0083] The negatively charged polymerizable phospholipids, chelated withmetal ions such as Ca²⁺, can be formed into water-free liposomes andconverted into spherical bilayer liposomes by exposure to chelatingagents. Additionally, negatively charged polymerizable phospholipids canbe mixed with the novel fatty acids and targeted fatty acids describedabove, and water-free composite structures can be formed in the presenceof divalent cations. Following conversion of water-free liposomes tospherical liposomes with internal aqueous space in the presence ofchelating agents, resulting liposomes can be cross-linked forstabilization by polymerization initiators in the same manner as for thenon-charged liposomes described above.

[0084] Thus, in one embodiment, the present invention encompassespolymerized liposomes which comprise (a) one or more of the fatty acidsdescribed above which may optionally be substituted with a suitablepolymer spacer, activated linker, and one or more lectin targetingmolecules; (b) one or more of the negatively charged polymerizablelipids described herein; (c) one or more non-polymerizable fatty acidsor phospholipids; and (d) optionally cholesterol. Preferably, thepolymerized liposomes comprises from about 0% to about 15% fatty acidand about 0% to about 100% negatively charged polymerizable lipids; andabout 0% to about 50% non-polymerizable fatty acids, phospholipids orcholesterol; for example, DODPC/DODPG/Targeted fattyacid/cholesterol/DSPC.

[0085] Formulations/Compositions

[0086] The polymerized liposomes and targeted polymerized liposomes ofthe present invention are used as the carriers in a drug deliverysystem, especially an oral drug delivery system. The present inventionthus also encompasses therapeutic formulations and compositions usingthese polymerized liposomes. The following describes representativematerials which can be encapsulated in the liposomes of the presentinvention to form therapeutic compositions, methods of encapsulatingthose materials, and modes of administering the therapeutic compositionsto a patient.

[0087] Materials to be Encapsulated

[0088] The polymerized liposomes of the present invention have utilityfor the oral and/or mucosal delivery of vaccines, antigens, allergens,diagnostic agents therapeutic agents and drugs. The polymerizedliposomes of the present invention may be designed to carry a widevariety of antigens including, but not limited to diphtheria toxoid,influenza hemeagglutinin, ospA antigen from Lyme disease bacterium, andHTLV envelope protein antigen. Antigens to poliovirus, rhinovirus,rabies, vaccinia, Epstein-Barr virus, hepatitis, HTLV, herpes virus andhuman immunodeficiency virus are just examples of the many types ofantigens which may be encapsulated into the liposomes of the presentinvention.

[0089] The polymerized liposomes of the present invention can be usedfor the oral and/or mucosal delivery of a wide variety of therapeutics,including but not limited to, antineoplastic agents, antibiotics,antifungals, antimicrobials, vaccines, insulin, cytokines, interferon,hormones, calcitonin, fertility drugs, antiviral agents (ddi, AZT, ddc,acyclovir and the like), antibacterial agents, DNA and RNA nucleotides,i.e., useful for gene therapy.

[0090] As used herein, the term “biologically active substance” refersto eukaryotic and procaryotic cells, viruses, vectors, proteins,peptides, nucleic acids, polysaccharides and carbohydrates, lipids,glycoproteins, and combinations thereof, and synthetic organic andinorganic drugs exerting a biological effect when administered to ananimal. For ease of reference, the term is also used to includedetectable compounds such as radiopaque compounds including air andbarium, magnetic compounds, fluorescent compounds, and radioactivecompounds. The active substance can be soluble or insoluble water.Examples of biologically active substances include anti-angiogenesisfactors, antibodies, antigens, growth factors, hormones, enzymes, anddrugs such as steroids, anti-cancer drugs or antibiotics, as well asmaterials for use as insecticides or insect repellents, fertilizers andvitamins, or any other material having a biological effect wherecontrolled release is desirable.

[0091] In a diagnostic embodiment, the polymerized liposome incorporatesa pharmaceutically acceptable gamma-emitting moiety, including but notlimited to, indium and technetium, magnetic particles, radiopaquematerials such as air or barium and fluorescent compounds.

[0092] Encapsulation of Biologically Active Material

[0093] Materials are generally incorporated into the liposomes at thetime of formation, following polymerization using sonication of asolution of the material which contains the liposomes, and followingpolymerization by rehydration of a thin film of the liposomes.

[0094] The following is a general method for the preparation ofpolymerized liposomes wherein a biologically active substance isentrapped prior to the polymerization of the monomeric polymerizableliposome. First, the monomeric liposome can be prepared by the thin filmhydration of polymerizable phospholipids and fatty acid mixture asdescribed above. To form the thin film, a monomeric phospholipid and,optionally, a polymerizable fatty acid or targeted polymerizable fattyacid, is dissolved, in a suitable organic solvent such as chloroform,and the solution is then dried to form a thin film of phospholipid.Alternatively, a mixture of polymerizable phospholipid, fatty acids andtargeting reagents is dissolved in tertiary butanol. The mixture islyophilized to create a powdery substance that can be easily rehydratedfor liposome generation and entrapment of payload drug or protein. Asolution containing substance to be entrapped is added. At this stage,it is preferable to establish an inert atmosphere. The lipid film isthen hydrated by gently shaking and sonicating the solution at atemperature of from about 30 to 50° C., usually around 40° C., forbetween five minutes and two hours, preferably around five minutes. Oncethe lipid film is hydrated, the trap ratio of the liposome can beincreased by performing one or more freeze-thaw cycles on the liposomesolution. This is particularly useful when the material beingincorporated is hydrophilic in nature. Next, the polymerization isinitiated in the presence of free radical initiators such as sodiumbisulfite and potassium persulfate at a temperature between 25° C. and40° C. until the polymerization is essentially complete. The desireddegree of polymerization is from 30 to 100 percent.

[0095] Unentrapped biologically active substance can be removed byseveral means, including repeated centrifugation, decantation, gelfiltration, and dialysis. The polymerized liposomes are then suspendedin a buffer solution. The buffer solution has a pH preferably between pH4.5 and pH 9.5, more preferably at physiological pH.

[0096] This method of entrapping biologically active substances ispreferred because it does not involve the use of organic solvents. Useof organic solvents can denature biologically active substances.Further, the temperature requirements are mild, with the temperaturetypically not exceeding 40° C.

[0097] Materials can be entrapped within the liposomes, as well as oralternatively in one or more of the lipid layers of the phospholipidbilayer. This is typically determined by thehydrophobicity/hydrophilicity of the material to be incorporated as wellas the method of preparation.

[0098] Modes of Administering the Polymerized Liposomes to a Patient

[0099] The polymerized liposomes of the present invention areadministered by those routes which optimize uptake by mucosa. Forexample, oral, sublingual, buccal, rectal, vaginal and intranasal arepreferred routes of administration. However, topical, transdermal andparenteral delivery may also be used. The most preferred route is oral.Further, the polymerized liposomes are particularly suitable fordelivery through mucosal tissue or epithelia. The polymerized liposomesof the invention can be delivered orally in the form of tablets,capsules, cachets, gelcaps, solutions, suspensions, creams, ointments,suppositories and the like. When the dosage unit form is a capsule, itcan contain, in addition to the material of the above type, a liquidcarrier or adjuvant, when the liposomes contain an antigen. Ifadministered topically the liposomes will typically be administered inthe form of an ointment or transdermal patch. If administeredintranasally the liposomes will typically be administered in an aerosolform, spray, mist or in the form of drops. Suitable formulations can befound in Remington's Pharmaceutical Sciences, 16th and 18th Eds., MackPublishing, Easton, Pa. (1980 and 1990), and Introduction toPharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia(1985), each of which is incorporated herein by reference.

[0100] The polymerized liposomes of the present invention are suitablefor administration to mammals, including humans, as well as otheranimals and birds. For example, domestic animals such as dogs and cats,as well as domesticated herds, cattle, sheep, pigs and the like may betreated or vaccinated with the polymerized liposomes of the presentinvention.

[0101] The polymerized liposomes of the present invention have use invaccine preparations. The preparation of vaccines containing animmunogenic polypeptide as the active ingredient is known to one ofskill in the art.

[0102] Vaccine Formulations

[0103] Suitable preparations of vaccines include liquid solutions orsuspensions; solid forms such as capsules and tablets, liquids forinjections, may also be prepared. The active immunogenic ingredients areoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the vaccine preparationmay also include minor amounts of auxiliary substances such as wettingor emulsifying agents, pH buffering agents, and/or adjuvants whichenhance the effectiveness of the vaccine.

[0104] Examples of adjuvants which may be effective, include, but arenot limited to: aluminum hydroxide, muramyl dipeptides, includingN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,saponins, saponin derivatives and fractions, QuilA, polymer adjuvants,including but not limited to block copolymers of polyethylene oxide andpolypropylene oxide, monophosphoryl lipid A, lipid A derivatives,cholera toxin or E. coli heat labile toxin, non-toxic mutants of choleratoxin or labile toxin.

[0105] The effectiveness of an adjuvant may be determined by measuringthe induction of antibodies or cellular immunity directed against animmunogenic polypeptide containing an antigenic epitope, the antibodiesresulting from administration of this polypeptide in vaccines which arealso comprised of the various adjuvants.

[0106] The polypeptides may be formulated into the vaccine as neutral orsalt forms. Pharmaceutically acceptable salts include the acid additionsalts (formed with free amino groups of the peptide) and which areformed with inorganic acids, such as, for example, hydrochloric orphosphoric acids, or organic acids such as acetic, oxalic, tartaric,maleic, and the like. Salts formed with free carboxyl groups may also bederived from inorganic bases, such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine and the like.

[0107] The vaccines of the invention may be multivalent or univalent.Multivalent vaccines are made from recombinant viruses that direct theexpression of more than one antigen.

[0108] Many methods may be used to introduce the vaccine formulations ofthe invention; these include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,rectal, and via scarification (scratching through the top layers ofskin, e.g., using a bifurcated needle).

[0109] The patient to which the vaccine is administered is preferably amammal, most preferably a human, but can also be a non-human animalincluding but not limited to cows, horses, sheep, pigs, fowl (e.g.,chickens), goats, cats, dogs, hamsters, mice and rats.

[0110] The vaccine formulations of the invention comprise an effectiveimmunizing amount of the antigenic protein and a pharmaceuticallyacceptable carrier or excipient. Vaccine preparations comprise aneffective immunizing amount of one or more antigens and apharmaceutically acceptable carrier or excipient. Pharmaceuticallyacceptable carriers are well known in the art and include but are notlimited to saline, buffered saline, dextrose, water, glycerol, sterileisotonic aqueous buffer, and combinations thereof. One example of suchan acceptable carrier is a physiologically balanced culture mediumcontaining one or more stabilizing agents such as stabilized, hydrolyzedproteins, lactose, etc. The carrier is preferably sterile. Theformulation should suit the mode of administration.

[0111] The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. The compositioncan be a liquid solution, suspension, emulsion, tablet, pill, capsule,sustained release formulation, or powder. Oral formulations can includestandard carriers such as pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, etc.

[0112] Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water-free concentrate in a hermetically sealed container suchas an ampoule or sachet indicating the quantity of active agent. Wherethe composition is administered by injection, an ampoule of sterilediluent can be provided so that the ingredients may be mixed prior toadministration.

[0113] The precise dose of vaccine preparation to be employed in theformulation will also depend on the route of administration, and thenature of the patient, and should be decided according to the judgmentof the practitioner and each patient's circumstances according tostandard clinical techniques. An effective immunizing amount is thatamount sufficient to produce an immune response to the antigen in thehost to which the vaccine preparation is administered.

[0114] Use of purified antigens as vaccine preparations can be carriedout by standard methods. For example, the purified protein(s) should beadjusted to an appropriate concentration, formulated with any suitablevaccine adjuvant and encapsulated within the polymerized liposome.Suitable adjuvants may include, but are not limited to: mineral gels,e.g., aluminum hydroxide; surface active substances such as lysolecithinor pluronic polyols; polyanions; peptides; oil emulsions; alum, and MDP.The immunogen may also be incorporated into liposomes, or conjugated topolysaccharides and/or other polymers for use in a vaccine formulation.In instances where the recombinant antigen is a hapten, i.e., a moleculethat is antigenic in that it can react selectively with cognateantibodies, but not immunogenic in that it cannot elicit an immuneresponse, the hapten may be covalently bound to a carrier or immunogenicmolecule; for instance, a large protein such as serum albumin willconfer immunogenicity to the hapten coupled to it. The hapten-carriermay be formulated for use as a vaccine.

[0115] Effective doses (immunizing amounts) of the vaccines of theinvention may also be extrapolated from dose-response curves derivedfrom animal model test systems.

[0116] The present invention thus provides a method of immunizing ananimal, or treating or preventing various diseases or disorders in ananimal, comprising administering to the animal an effective immunizingdose of a vaccine encapsulated within a polymerized liposomes of thepresent invention.

[0117] Certain embodiments of the invention are illustrated, and notlimited, by the following working examples.

EXAMPLE 1 (A) Synthesis of 2,4-ODPEG

[0118] 2,4-ODPEG was synthesized as follows. 50 mg of2,4-octadecadienoic acid (2,4-OD) was dissolved in 5 ml ofN,N-dimethylformamide (DMF) under inert gas (argon), with constantstirring. 1.78 g of polyethylene glycol (average molecular weight of400) and 217 mg of dimethylaminopyridine (DMAP) were added. Then 410 mgof 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDCI) was added,and the solution stirred at room temperature for 24 hours. The reagentvessel was kept wrapped in aluminum foil during this time, to protectagainst UV degradation. The reaction was then quenched by the additionof a few milliliters of ice water, to destroy remaining EDCI. DMF andwater were then evaporated under vacuum.

[0119] The compound thus obtained was purified in two stages. First, itwas purified by dialysis against distilled water (500 ml) in aSpectraphor® dialysis tube (MWCO 500). The dialysis procedure wasrepeated a total of four times. The solution was evaporated under vacuumand then further purified by reversed-phase chromatography. The column(RP18) was first washed with water, then the fraction containing ODPEGwas eluted using a 4:1 ethanol:water eluent. The fraction collected wasevaporated under vacuum, to give a 53% yield of 2,4-ODPEG. The 2,4-ODPEGproduct was analyzed by thin layer chromatography (TLC) and byultraviolet (UV) absorption spectroscopy (λ_(max)=255 nm).

(B) Synthesis of 2,4-ODPEG

[0120] 110 mg of 2,4-octadecadienoic acid (2,4-OD) (NOF Corp., Japan),61 mg of dimethylaminopyridine (DMAP, Aldrich) and 0.84 g ofpolyethylene glycol (Averg. M.W. 400, Aldrich) were dissolved in 7 ml ofdistilled methylene chloride under argon gas with constant stirring.Then, 72 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Hcl (EDCl)(Aldrich) was added (cooling if necessary), and the solution was stirredat room temperature for 24 hours. The reagent vessel was kept wrapped inaluminum foil during this time. The reaction was then quenched by theaddition of a few milliliters of water. The organic solvent was removedunder reduced pressure.

[0121] The compound thus obtained was purified in two stages. First, itwas purified by dialysis against distilled water (500 ml×4) in aSpectra/Por dialysis tube (MWCO 500). The inner solution was lyophilizedand the resulting white solid was further purified by reversed-phasecolumn chromatography. The column (RP-18, EM Science) was eluted withwater followed by a 4:1 ethanol:water eluent. The fraction collected wasconcentrated then lyophilized to give 207 mg (80% yield) of ahygroscopic white solid product with λ_(max)=266 nm.

(C) Synthesis of 2,4-ODPEGSu

[0122] The 2,4-ODPEG succinic acid derivative (2,4-ODPEGSu) was thenprepared by reacting 2,4-ODPEG with succinic anhydride. First, 41 mg of2,4-ODPEG and 25 mg of succinic anhydride (Aldrich) were dissolved in 2ml of methylene chloride. 8.3 mg of DMAP was added, and the reaction wasstirred at room temperature, under nitrogen gas protection and with thevessel tightly sealed and wrapped in aluminum foil, for 24 hours. Thereaction was quenched by addition of a few milliliters of ice water,then evaporated to dryness under vacuum. The resulting product waspurified first by dialysis and reversed phase chromatography, asdescribed above, then by a second dialysis stage (MWCO 1000). Theproduct was freeze-dried, and kept at −20° C. The yield of 2,4-ODPEGSuobtained from 2,4-ODPEG in this example was quantitative, so that thetotal synthesis of the polymerizable fatty acid has a yield, in thisexample, of about 53%.

EXAMPLE 2 Coupling of 2,4-ODPEGSu with EEA

[0123] A polymerizable fatty acid was coupled to a lectin, and thedegree of lectin modification determined, according to the followingmethod.

[0124] Materials and Methods

Coupling of 2,4-ODPEGSu with EEA

[0125] EEA was coupled to 2,4-ODPEGSu by the following method. 0.5 mg of2,4-ODPEGSu was dissolved in 0.6 ml of a pH 5.4 MES buffer(2-(N-morpholino)ethane-sulfonic acid, sodium salt) (Aldrich). 3 mg ofN-octylglucoside, 5 mg of EDCI, and 11.5 mg ofN-hydroxylsulfosuccinimide (NHS) were added. The reaction was run atroom temperature for 5 minutes, with constant stirring. Next, 1.46 mg ofEEA in a phosphate buffered saline (PBS) solution, and 0.5 ml of 1 molarHEPES buffer were added. The pH of the solution was adjusted to 7.6, andthe solution was stirred at 4° C. overnight. The product was dialyzedagainst 0.2 mM PBS (300 ml×5) in a Spectra Por dialysis tube(MWCO=2,000) for a total of 30 hours. The purified product wastransferred to a vial and kept at 4° C. until further use. Other lectinscan be linked to 2,4-ODPEGSu by following a similar procedure.

[0126] Determination of the Degree of Lectin Modification

[0127] The degree of lectin modification is determined as follows. Fivestock solutions are prepared:

[0128] Solution A: 0.1M Na₂B₄O₇ in 0.1M NaOH

[0129] Solution B: 0.1M NaH₂PO₄

[0130] Solution C: 0.1M Na₂SO₃ (prepared within 48 hours)

[0131] Solution D: 10 ml of Solution B+0.15 ml of Solution C

[0132] Solution E: 50 ml of TNBS in 5 ml distilled water

[0133] where TNBS is 2,4,6-trinitro-benzenesulphonic acid (SIGMA). 0.685ml of the 2,4-ODPEGSu-EEA solution (protein concentration of 0.292mg/ml) is diluted with solution A to a final volume of 1 ml. To this isadded 100 μl of solution E and mixed vigorously. The resulting solutionis incubated at 40° C. for 45 minutes, then the reaction is stopped byaddition of 1 ml of solution D. A standard solution is prepared bymixing 100 μl of solution E in 1 ml of solution A and 1 ml of solutionD. The degree of lectin modification is then calculated from theabsorbance of the solution at 420 nm. The degree of modification in thisexample is 51%.

EXAMPLE 3 Polymerizable Liposomes Incorporating Membrane Proteins

[0134] DODPC is dissolved in octylglucoside, and the desired peptide isdissolved in PBS. The peptide solution is then added to the lipid insideof a dialysis membrane (MWCO=10,000) and dialyzed versus PBS (2 mlversus 1 liter) for approximately four hours, with two changes of PBS.The liposomes spontaneously form in the dialysis membrane. The liposomesthus formed can be polymerized using a redox couple initiator (e.g.,NaHSO₃ and K₂S₂O₈).

EXAMPLE 4 Polymerized Liposomes

[0135] A mixture of lipids containing a polymerizable lipid can belyophilized to form a powdery material, then rehydrated with a solutioncontaining the desired peptides or antigens to be encapsulated in theliposome or to be incorporated into the lipid bilayer. The rehydratingtakes place at a temperature above the melting point of the lipidmixture. The resulting liposomes are then sonicated at 45° C. forapproximately 5 minutes to reduce the size of the liposomes to aroundthe 200 nm range. The liposomes are then polymerized using any knowntechnique. The free peptide can be separated from the liposomes bycentrifuging the liposomal preparation at approximately 100,000×g.Alternatively, the polymerized liposome solutions can be passed throughan ultrafiltration column to purify and concentrate the liposomescontaining peptide.

[0136] A preferred method uses a mixture of 2,4-DODPC andlectin-modified 2,4-ODPEGSu as the polymerizable lipid mixture,dissolved in tert-butanol. After rehydration with a solution of thedesired peptide or antigen, the liposomes are sonicated and polymerizedusing a sodium bisulfite (580 μM) potassium persulfate (127 μM) redoxcouple initiator.

EXAMPLE 5 Measurement of the Absorption of Biologically ActiveSubstances Entrapped in Polymerized Liposomes

[0137] Polymerized liposomes containing ¹²⁵I-BSA can be orallyadministered to rats. The absorption of ¹²⁵I-BSA into the blood can thenbe examined. ¹²⁵I-BSA containing monomeric liposomes and ¹²⁵I-BSAsolution are used as controls. The polymerized liposomes are prepared asdescribed infra.

[0138] Each formulation, including the control ¹²⁵I-BSA solution, isadministered intragastrically with a ball-tipped needle and blood issampled at appropriate intervals from the tail vein. To distinguishbetween transport of ¹²⁵I-BSA in the context of liposomes, free ¹²⁵I-BSAand the radiolabelled degradation product of ¹²⁵I-BSA, the blood samplesare separated into three fractions: 1) cell debris fraction, 2)trichloroacetic acid (TCA) precipitable fraction, and 3) TCAnon-precipitable fraction.

[0139] Feces of rats are homogenized with water and centrifuged toseparate solids. Radioactivity in the whole homogenate and sedimentedsolid are then compared. In the case of polymerized liposomeadministered rats, the difference in the amount of total radioactivityobserved in the solid, compared with the amount from monomeric liposomeadministered rats, shows the relative stability of polymerized liposomesin the G-I tract.

[0140] Because elimination of the precipitable fraction in blood afterintravenous injection can be slow, the TCA non-precipitable fraction issmaller in animals administered material in polymerized liposomes, ascompared to material administered in conventional liposomes andsignificantly less than when material is administered in solution.

[0141] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

[0142] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

What is claimed is:
 1. A polymerizable compound of the formula:R₄—X-PEG-Y—B wherein R₄ is a lipophilic chain containing at least onepolymerizable moiety; X and Y are either or both not present or areeither or both represent a chemically stable bond or linker moiety; B is—O—C(O)—(CH₂)_(p)CO₂H, C(O)NHR₆, C(O)NH₂, or an aldehyde wherein p is aninteger form 0 to 12 and R₆ is a C₁ to C₅ alkyl group; and PEG is apolyethylene glycol group having an average molecular weight from about200 to about 2000 g/mol; or a pharmaceutically acceptable salt thereof.2. The polymerizable compound of claim 1 wherein said R₄ is a lipophilicchain containing one or more double bonds, triple bonds or thiol groupsor combinations thereof.
 3. The polymerizable compound of claim 1wherein said X and Y are independently selected from the groupconsisting of an ether bond, an amide bond or an ester bond.
 4. Thepolymerizable compound of claim 1 wherein said B is a carboxylic acidgroup or an NH₂ group.
 5. The polymerizable compound of claim 1 whereinsaid lipophilic group is an alkyl group containing from 12 to 30 carbonatoms.
 6. The polymerizable compound of claim 1 which further comprisesa targeting ligand covalently bound to said B moiety.
 7. Thepolymerizable compound of claim 6 wherein said targeting ligand is alectin.
 8. The polymerizable compound of claim 7 wherein said lectin isEEA, FITC-EEA, UEA-I or WGA.
 9. A polymerized liposome which comprisesfrom about 1 to about 50% polymerizable fatty acids of claim 1 or
 6. 10.The polymerized liposome of claim 9 which further comprises a vaccine,an antigen, or a biologically active drug and optionally cholesterol.11. A polymerizable fatty acid of the formula:CH₃—(CH₂)₁₂—CH═CH—CH═CH—C(O)—(OCH₂CH₂)_(n)—O—C(O)—CH₂—CH₂—CO₂H wherein nis an average number of —OCH₂CH₂— units from about 4 to about 45, or apharmaceutically acceptable salt thereof.
 12. The fatty acid of claim 11wherein n is an average number of —OCH₂CH₂— units from about 6 to about12.
 13. A polymerizable fatty acid according to claim 11 which furthercomprises a targeting ligand covalently bound to said polymerizablefatty acid.
 14. The polymerizable fatty acid of claim 13 wherein saidtargeting ligand is a lectin.
 15. The polymerizable fatty acid of claim14 wherein said lectin is UEA, WGA, EEA or FITC-EEA.
 16. A polymerizedliposome which comprises one or more polymerizable lipids and apolymerizable fatty acid of the formula:CH₃—(CH₂)₁₂—CH═CH—CH═CH—C(O)—(OCH₂CH₂)_(n)—O—C(O)—CH₂—CH₂—CO₂H wherein nis an average number of —OCH₂CH₂— units from about 6 to about
 12. 17. Apolymerized liposome which comprises one or more polymerizable lipidsand a polymerizable fatty acid covalently bonded to a targeting ligand,the polymerizable fatty acid having the formula:CH₃—(CH₂)₁₂—CH═CH—CH═CH—C(O)—(OCH₂CH₂)_(n)—O—C(O)—CH₂—CH₂—CO₂H wherein nis average number of —OCH₂CH₂— units from about 6 to about
 12. 18. Apolymerized liposome according to claim 17 wherein the targeting ligandis a lectin.
 19. A polymerized liposome according to claim 18 whereinthe lectin is EEA, FITC-EEA, UEA or WGA.
 20. A polymerized liposomeaccording to claim 16 or 17 wherein the polymerizable lipid is apolymerizable phospholipid.
 21. A polymerized liposome according toclaim 16 or 17 which further comprises a non-polymerizable compound. 22.A polymerized liposome according to claim 21 wherein thenon-polymerizable compound is cholesterol.
 23. A polymerized liposomeaccording to claim 16 or 17 which further comprises a vaccine, anantigen or a biologically active drug.
 24. A polymerizable phospholipidhaving the structure:

wherein at least one of R, R′ and R″ is a polymerizable group and atleast one of the remaining groups is selected from the group consistingof phosphoryl inositol, phosphoryl glycerol and phosphoryl serine.
 25. Apolymerizable phospholipid according to claim 24 wherein at least one ofR, R′ and R″ is a 2,4-octadecadienoyl group.
 26. A polymerized liposomeuseful for oral drug delivery which comprises: (a) a polymerizable fattyacid of claim 1; and (b) a polymerizable phospholipid of claim
 24. 27.The polymerized liposome of claim 26 which further comprisescholesterol.
 28. The polymerized liposome of claim 26 which furthercomprises a polymerizable fatty acid of claim
 11. 29. The polymerizableliposome of claim 28 which further comprises cholesterol.
 30. Apolymerized liposome useful for oral drug delivery which comprises: (a)a polymerizable fatty acid of claim 11; and (b) a polymerizablephospholipid of claim
 24. 31. The polymerized liposome of claim 30 whichfurther comprises cholesterol.
 32. The polymerized liposome of claim 30which further comprises a lectin.
 33. The polymerized liposome of claim26 or 30 which further comprises a vaccine.